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We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as β-thalassemia and sickle cell disease.

Islet transplantation is a promising treatment for selected patients with type 1 diabetes mellitus (T1DM). Hypoxia and oxidative stress are major causes of damage to transplanted islets. Mesenchymal stem cells (MSCs) have been shown to enhance cell survival mainly through paracrine secretion. However, mechanisms of action underlying the protective effects of MSCs on islets have not been fully elucidated. In this study, we investigated whether human umbilical cord-derived MSCs (huc-MSCs) could inhibit hypoxia and ROS-related cell death of neonatal porcine islet cell clusters (NICCs) and further determined the underlying molecular mechanisms. NICCs were cultured in vitro under normoxic and hypoxic (1% O2) conditions with or without MSC-conditioned medium (MSC-CM). Apoptosis of NICCs was evaluated by the AO/EB staining and Annexin V/PI flow cytometry analysis. Total and mitochondrial ROS production was detected by fluorometric assays. Western blot and the ERK pathway inhibitor, PD98059, were used to assess the possible pathways involved. The results showed that MSC-CM suppressed hypoxia-induced oxidative stress and cell death of NICCs. MSC-CM also activated several pro-survival pathways in NICCs under hypoxic conditions. Furthermore, MSC-secreted exosomes and IL-6 partially recapitulated the multifunctional benefits of MSC-CM. This study showed that huc-MSCs protected NICCs from hypoxia-induced cell death by regulating the cell redox state and cell signaling pathways. This increased understanding may enable MSCs to become a more promising adjuvant cell therapy for islet transplantation. Impact statement The utilization of mesenchymal stem cells (MSCs) is a promising approach to serve as adjuvant therapy for islet transplantation. But the inability to translate promising preclinical results into sound therapeutic effects in human subjects indicates a lack of key knowledge of MSC-islet interactions that warrant further research. Hypoxia and oxidative stress are critical factors which lead to a tremendous loss of islet grafts. However, previous studies mainly focused on other aspects of MSC protection such as inducing revascularization, enhancing insulin secretion, and reducing islet apoptosis. In this study, we aim to investigate whether MSC can protect islet cells from hypoxic damage by inhibiting ROS production and the potential underlying pathways involved. We also explore the effects of MSC-derived exosomes and IL-6 on hypoxia-injured islets. Our data provide new molecular targets for developing MSC applications, and this may ultimately promote the efficiency of clinical islet transplantation.

Background The dysfunction of islet grafts is generally attributed to hypoxia-induced damage. Mesenchymal stem cells (MSCs) are currently thought to effectively protect cells from various risk factors via regulating autophagy. In our study, we investigated if human umbilical cord-derived MSCs could ameliorate hypoxia-induced apoptosis in porcine islets by modulating autophagy, and we explored the underlying mechanisms. Methods Neonatal porcine islet cell clusters (NICCs) were cultured with human umbilical cord-derived MSC conditioned medium (huc-MSC-CM) and RPMI-1640 medium (control) under hypoxic conditions (1% O2 ) in vitro. NICCs were treated with 3-methyladenine (3-MA) and chloroquine (CQ) to examine the role of huc-MSC-CM in regulating autophagy. Finally, the levels of several cytokines secreted by huc-MSCs were detected by ELISAs, and the corresponding inhibitors were applied to investigate which cytokine mediates the protective effects of huc-MSC-CM. The effects of huc-MSC-CM on NICCs viability and autophagy were examined using AO/PI staining, flow cytometry analysis, transmission electron microscopy (TEM) and confocal fluorescence microscopy analysis. The insulin secretion of NICCs was tested with an insulin immunoradiometric assay kit. Results Compared to the control, the huc-MSC-CM treatment improved the viability of NICCs, inhibited apoptosis, increased autophagic activity and the levels of PI3K class III and phosphorylated Akt, while the ratio of phosphorylated mTOR/mTOR was reduced. These changes were reversed by CQ and 3-MA treatments. High concentrations of IL-6 were detected in hu-MSC-CM. Furthermore, recombinant IL-6 pre-treatment exerted similar effects as huc-MSC-CM, and these effects were reversed by a specific inhibitor of IL-6 (Sarilumab). Conclusions Our results demonstrated that huc-MSC-CM improved islet viability and function by increasing autophagy through the PI3K/Akt/mTOR pathway under hypoxic conditions. Additionally, IL-6 plays an important role in the function of huc-MSC-CM.

Atherosclerosis (AS) is a progressive disease characterized by endothelial injury and lipid aggregation in the arterial walls. Studies have reported that endothelial progenitor cells (EPCs) derived from the bone marrow (BM) might provide an endogenous repair mechanism by differentiating into endothelial cells to replace the dysfunctional endothelium. Our study aims to investigate the effect of EPCs derived from rat BM on AS. EPCs transduced by recombinant adeno-associated virus-green fluorescent protein (GFP) were transplanted into a rat AS model. After 2 months of transplantation, the localization of GFP-labeled cells, morphology, and lipid content in the aorta were examined. GFP-labeled EPCs were found in the endothelial monolayer of the artery vessel in the GFP/EPC group. Hematoxylin and eosin staining suggested that the lipid deposits in the aortic endothelium in the EPC/GFP group were less compared with those in the untreated group. Oil Red O staining of liver slices showed that lipid droplets were obviously decreased in the GFP/EPC group. The endothelial nitric oxide synthase and apolipoprotein E mRNA levels in the GFP/EPC group were significantly higher, but the intercellular cell adhesion molecule-1 mRNA level was significantly lower compared with the control group. The results suggest that EPCs derived from the BM can repair the injured endothelium and promote an atherosclerotic lesion regression. Therefore, EPCs may provide a useful tool for the treatment of AS.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Background Hypoxia-induced damage is one of the key factors associated with islet graft dysfunction. Mesenchymal stem cells (MSCs) could be used to enhance the therapeutic effect of islet transplantation due to their paracrine potential such as exosomes. In this study, we investigated whether exosomes from human umbilical cord-derived MSC-conditioned medium (hu-MSC-CM) could increase the survival and function of neonatal porcine islet cell clusters (NICCs) exposed to hypoxia. Methods Neonatal porcine islet cell clusters were cultured with hu-MSC-CM, with or without exosomes, and native medium RPMI-1640 (Control) under hypoxic conditions (1% O2 ). The effects of exosomes on NICCs viability and function in vitro were examined by FACS, the Loops system, and the Extracellular Flux assay, respectively. Results Compared with NICCs cultured in RPMI-1640 medium and hu-MSC-CM without exosomes, the survival ratio, viability, and function increased in NICCs cultured in hu-MSC-CM with exosomes. Conclusions This study found that hu-MSC-CM could protect NICCs from hypoxia-induced dysfunction, and exosomes played an important role in hypoxic resistance, suggesting a potential strategy to improve islet transplantation outcomes.

We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1AFig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1BFig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authorsView Large Image | Download PowerPoint Slide

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol . 72:1593–1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol . 72:9835–9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused ≈40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

The human embryonic kidney cell line, HEK293, is readily transduced by the most commonly used AAV2 serotype vectors because these adherent cells abundantly express the cell surface receptor, heparan sulfate proteoglycan (HSPG), and one of the co-receptors, human fibroblast growth factor receptor 1 (FGFR1) for AAV2. However, a human erythroleukemia cell line, K562, commonly used as a model for human hematopoietic cell transduction, is not transduced as efficiently, although these cells grown in suspension, express both HSPG and FGFR1 only at modestly lower levels. We reasoned that the lack of proximity of HSPG and FGFR1 on K562 cells might account for the suboptimal transduction of these cells, and hypothesized that if the transduction was performed at high cell density, presumably allowing for HSPG on one cell to come in close proximity to FGFR1 on the neighboring cell, then AAV2 bound to HSPG on one cell could utilize FGFR1 on the neighboring cell to gain entry in the latter, and vice versa, thus leading to increased transduction. To test this hypothesis, HEK293 and K562 cells were transduced with 3×103 vgs/cell of scAAV2-EGFP vectors either at low-density (60,000 cells in 50 ml) or at high-density (480,000 cells in 50 ml). Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. Whereas ~90% of HEK293 cells expressed the transgene at low-density, the transduction efficiency at high-density increased up to ~95%. On the other hand, only ~35% of K562 cells were transduced at low-density, but interestingly, the transduction efficiency at high-density increased to ~65%, thus corroborating that close cell-cell contact facilitated vector entry into these cells. That the initial cell-cell contact was critical in achieving improved transduction, was further corroborated by experiments in which cells were transduced at low-density, and subsequently pooled together to reach high-density, and conversely, cells were transduced at high-density, and soon after transduction, were diluted to low-density. The increased transduction was observed only under the latter condition. Similar results were obtained with two additional human cell lines, M07e and Raji, which express low to extremely low levels of HSPG and FGFR1, respectively, and consequently, are transduced extremely poorly by AAV2 vectors. These studies were also extended to include primary human CD34+ hematopoietic stem/progenitor cells (HSPCs), also grown in suspension, and known to be transduced sub-optimally by AAV2 vectors. In these studies also, whereas only~5% of these cells were transduced at low-density, the transduction efficiency increased up to ~20% at high-density. Taken together, our studies have revealed a novel mechanism, which we have termed “cross-transduction”, which AAV vectors exploit to gain entry into target cells, and which may have implications in the optimal use of AAV vectors in human gene therapy applications.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). However, the transduction efficiency of these vectors ranged between ~6-87% in HSPCs obtained from several different donors (n=11). Such a wide range of transduction efficiency of AAV6 vectors is presumably due to different levels of expression of the putative receptors and/or co-receptors on these cells. In our more recent studies, we observed that the transduction efficiency of AAV2 vectors could be augmented both by performing transduction of HSPCs with the wild-type (wt)-AAV2 vectors at high cell density, or by using capsid-modified Y444F+Y500F+Y731F+T491V-mutant AAV2 vectors. In the present studies, we examined whether similar strategies could also be employed to increase the transduction efficiency of HSPCs from donors that are not transduced efficiently by wt-AAV6 vectors. Primary human HSPCs were transduced with either 3×103 or 3×104 vgs/cell of scAAV6-EGFP vectors either at low-density (20,000 cells in 50 ml) or at high-density (200,000 cells in 50 ml). scAAV2-EGFP vectors were also used for comparison. Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results showed that whereas only ~14% of the cells transduced at low-density with high moi expressed the transgene, the transduction efficiency at high-density increased up to ~20% and 25%, at low, and high mois, respectively, also with a significant increase in the mean fluorescence intensity, thus corroborating that the initial cell-cell contact was a critical factor in achieving increased transduction. Next, the transduction efficiencies of the wt- and the capsid-modified triple-mutant (Y705F+Y731F+T492V) AAV6 vectors were compared. Again, the wt- and the capsid-modified quadruple-mutant (y444F+Y500f+Y731F+T491V) AAV2 vectors were used for comparison. HSPCs were transduced with 1×104 vgs/cell at high-density (200,000 cells in 50 ml), and transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results also clearly documented that the ~27% transduction efficiency of the wt-AAV6 vectors was increased by up to ~45% with the capsid-modified AAV6 vectors, with a concomitant increase in the mean fluorescence intensity. Taken together, these studies further corroborate the novel mechanism of “cross-transduction” by recombinant AAV vectors of human cells in general, and HSPCs in particular. Additional human HSPC transplant studies in a murine xenograft model in vivo are currently underway, the successful completion of which is likely to have significant implications of these strategies in the optimal use of AAV6 vectors in the potential gene therapy of human disorders involving the hematopoietic system.

Background aims Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. Results We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro . We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo . Conclusions These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. Disclosures Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.

Atherosclerosis (AS) is a progressive disease characterized by endothelial injury and lipid aggregation in the arterial walls. Studies have reported that endothelial progenitor cells (EPCs) derived from the bone marrow (BM) might provide an endogenous repair mechanism by differentiating into endothelial cells to replace the dysfunctional endothelium. Our study aims to investigate the effect of EPCs derived from rat BM on AS. EPCs transduced by recombinant adeno-associated virus-green fluorescent protein (GFP) were transplanted into a rat AS model. After 2 months of transplantation, the localization of GFP-labeled cells, morphology, and lipid content in the aorta were examined. GFP-labeled EPCs were found in the endothelial monolayer of the artery vessel in the GFP/EPC group. Hematoxylin and eosin staining suggested that the lipid deposits in the aortic endothelium in the EPC/GFP group were less compared with those in the untreated group. Oil Red O staining of liver slices showed that lipid droplets were obviously decreased in the GFP/EPC group. The endothelial nitric oxide synthase and apolipoprotein E mRNA levels in the GFP/EPC group were significantly higher, but the intercellular cell adhesion molecule-1 mRNA level was significantly lower compared with the control group. The results suggest that EPCs derived from the BM can repair the injured endothelium and promote an atherosclerotic lesion regression. Therefore, EPCs may provide a useful tool for the treatment of AS.

Cholesterol efflux is the first step in the reverse cholesterol transport (RCT) pathway, removing excess cholesterol from tissues, including the arterial wall, thus preventing the development of atherosclerosis. Adeno-associated virus (rAAV) has demonstrated significant promise as a DNA-delivery vector to treat serious human diseases. In this study, we constructed recombinant adeno-associated viruses coexpressing apoAI and SR-BI successfully, the double gene mRNA and protein were both strongly expressed in transduced HepG2 cells. A novel safe and efficient method of promoting the reverse cholesterol transport (RCT) may be established. These results may provide a new method for gene therapy of Arteriosclerosis.

Endothelial dysfunction/loss is a key event in the development of vascular diseases, including native atherosclerosis (AS). Recent studies have shown that endothelial progenitor cells (EPCs) have the ability to repair endothelial cells that have been lost or damaged following AS. As a result, the therapy of transplanting EPCs is a promising option for the treatment of AS. However, the therapeutic effect on AS with only EPCs transplantation has not been satisfactory. The upregulation of those genes, which prevent the progress of AS in EPCs, is a novel therapeutic strategy for AS. Because it can reduce macrophage foam cell formation and protect endothelial cells from the oxidation of low-density lipoprotein (ox-LDL), paraoxonase-1 (PON1) gene is a candidate for gene therapy in AS. In this study, a recombinant adeno-associated virus (rAAV) vector carrying the human paraoxonase-1 (hPON1) gene (rAAV-PON1) was constructed, and endothelial progenitor cells (EPCs) transfected with rAAV-PON1 were transplanted into the atherosclerosis model of Sprague-Dawley rats (SD rats). The results of doppler ultrasound and histological analysis showed that the group transplanted with the hPON1 gene-transfected EPCs was superior to the group transplanted only with the EPCs and was also better than the group with hPON1 gene injection alone. The results indicated that rAAV-mediated hPON1 gene-transfected EPCs is a potentially valuable new tool in the treatment of atherosclerosis.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.

Angiogenesis is a complicated process involving endothelial proliferation, migration, and finally formation of functional vessels, in which perivascular cells play assistant roles. Mesenchymal stem cells (MSCs) from distinct human tissues have been proved to share functional properties and gene-expression profile with perivascular cells and to be proangiogenic. In this study we compared the proangiogenic ability of three types of MSC derived from human adipose stromal cells (ADSCs), umbilical cord (UCMSCs) and endometrium (EMSCs). Three types of MSCs all located around the tubular like structures formed by EPCs in coculture system on Matrigel in vitro (Figure 1A). However, different MSCs showed different potential for stimulating vessel formation of EPCs. After 6 days' coculture, we found that EPCs seeded on ADSCs monolayer formed more organized capillary-like network (Figure 1B, P Furthermore, we compared their proangiogenic potential of three types of MSCs. Matrigel contained MSCs and EPCs (ratio 3:2, total 2*106) was injected into nude mouse dorsal subcutaneous. 7 days following the injection, Matrigel was stripped off for the analysis of microvessels. As shown in Figure 2, three types of MSCs all promoted the formation of new microvessels. ADSC had the strongest stimulating effects, and formed more microvessels containing a large number of erythrocytes as shown by the appearance and HE staining (6.43%±2.57%). However, microvessel structures were not observed in the matrigel containing EPCs alone. Moreover, we assessed the therapeutic effects of co-transplantation of EPCs and MSCs in mouse hindlimb ischemic model. Right femoral artery ligation and excision was performed on anesthetized nude mice. After 24h of surgery, EPCs and MSCs were co-transplanted by tail intravenous injection (n=5, total 6.25*105; MSC/EPC ratio, 3:2). Blood flow of the ischemic limb was measured at 0, 7, 14, 21, 28 days after cell transplantation using Laser Doppler Perfusion Imager (Figure 3A). The results showed that the co-transplantation of EPCs with ADSCs significantly improved blood flow of ischemic limb after day 7 of transplantation (0.94±0.10, P We queried whether the different stimulation effects of MSCs on the vessel formation of EPCs are caused by MSC-secreted proangiogenic cytokines. Firstly, we analyzed the effects of MSC-CM on the proliferation and migration of EPCs in vitro, and found that migration rates/numbers of EPCs was highly increased by ADSC-CM (69.52%±9.66%, P In conclusion, MSCs could promote the formation of new microvessles of EPCs, and ADSCs are more efficient than UCMSCs or EMSCs by secreting more proangiogenic cytokines and less inhibitor. Thus ADSCs may be an ideal cellular source for the treatment of ischemia vascular disease. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human beta-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human beta-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW(41/41) anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human beta-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human beta-globin gene sequences were present at approximately 1 copy per cell. The efficiency of the human beta-globin gene expression was determined to be up to 35% compared with the murine endogenous beta-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human beta-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human beta-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of beta-thalassemia and sickle-cell disease.

Abstract 4715 β -Thalassemia is one of the most common worldwide monogenic human diseases,caused by molecular defects in the human β -globin gene cluster leading to decrease or absence of β-globin. Loss of β -globin chains causes ineffective production of oxygen-carrying hemoglobin and therefore results in severe anemia. The treatment for β -Thalassemia major usually includes lifelong blood transfusions but chronic blood transfusion often causes iron overload, and accumulated iron produces tissue damage in multiple organs, so that iron chelating treatment is also needed. Bone marrow transplantation is another effective therapy, which can eliminate a patient's dependence on blood transfusions, however, it is difficult to find a matching donor for most patients; therefore it is only available for a minority of patients. Gene therapy is one potential novel therapy for treatment of inherited monogenic disorders. The long–term therapeutic strategy for this disease is to replace the defective β-globin gene via introduction of a functional gene into hematopoietic stem cells (HSCs). Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. AAV can infect both dividing and non-dividing cells and wild AAV integrates preferentially at a specific site on human chromosome 19. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome, mediating long-term and stable expression of the transgene. In this study, we used a hybrid rAAV6/2 vector carrying the human β-globin gene to transduce HSCs from a β -Thalassemia patient, followed by transplantation into irradiated BALB/c nude mice. One month post-transplantation, Hb was prepared from peripheral blood and analyzed by Western Blot and HPLC respectively. RNA and DNA were isolated from bone marrow cells (BMCs) from recipient mice transplanted with mock-infected or hybrid rAAV–globin-infected cells and analyzed by RT-PCR and PCR respectively. The results showed: 1. Human β-actin and β-globin transcripts were detected by RT-PCR in BMCs from all recipient mice, indicating that human HSCs were successfully transplanted in these mice and that the human β-globin gene was transcriptionally active in the donor cells. 2. The level of human hemoglobin expressed in peripheral red blood cells of recipient mice as measured by HPLC (ratio of β/α) was increased to 0.3 from 0.05 of pre-transplantation levels. Expression of human β-globin was also confirmed in recipient mice by Western Blot; a 2–3-fold increase compared with that of controls. Our results indicate that human HSCs from a β-Thalassemia patient can be efficiently transduced by a hybrid rAAV6/2-β-globin vector followed by expression of normal human β-globin protein. This study provides a proof-of-concept that rAAV6/2-mediated gene transfer into human HSCs might be a potential approach for gene therapy of β-Thalassemia. Disclosures: No relevant conflicts of interest to declare.

The transduction efficiency of adeno-associated virus 2 (AAV) vectors varies greatly in different cells and tissues. Whereas muscle and brain cells are transduced efficiently, controversies abound regarding hematopoietic cell transduction by AAV vectors. For human hematopoietic cells, we documented this problem to be related to the extent of AAV receptor expression (J. Virol., 71: 8262–8267, 1997). Murine hematopoietic cells express the receptor, yet are transduced poorly. Using a murine fibroblast cell line, we reported that the lack of transduction was due to inefficient intracellular trafficking of AAV from cytosol into the nucleus (J. Virol., 74: 992–996, 2000), and that treatment with hydroxyurea (HU) facilitated nuclear transport of AAV in these cells (J. Virol., 75: 4080–4090, 2001). In the present studies, we extended these observations to primitive murine hematopoietic cells. Murine c-kit+, lin− hematopoietic cells were transduced with recombinant AAV-lacZ vectors. Forty-eight hrs. post-transduction, nuclear and cytoplasmic fractions were isolated and low Mr DNA samples extracted from these fractions were analyzed on Southern blots. Approximately 85% of AAV genomes were present in the cytoplasmic fraction. However, when donor mice were injected intra-peritoneally with HU at 1 mg/g body weight, 24 hrs. prior to isolation of cells, the extent of AAV genomes detected in the cytoplasmic fraction was reduced to ~40%, with a corresponding increase in the nuclear fraction, which increased to ~60%, indicating that in vivo administration of HU facilitated nuclear transport of AAV. However, when DNA samples were electrophoresed on denaturing gels and analyzed on Southern blots, it was apparent that a significant fraction of the AAV genome present in the nuclear fraction from cells obtained from HU-treated mice was single-stranded. We tested the hypothesis whether single-stranded genomes were derived from virions that failed to undergo uncoating in the nucleus. Primitive hematopoietic cells, with and without HU-administration in vivo, were transduced with recombinant AAV-lacZ vectors, and 48-hrs. post-transduction, equivalent fractions were either mock-treated, or digested exhaustively with DNase I, and analyzed on DNA slot-blots using a lacZ probe. Whereas majority of the signal, which was resistant to DNase I, was detected in the cytoplasmic fraction in cells obtained from untreated mice, as expected, a substantial fraction of the signal in the nuclear fraction in cells obtained from HU-treated mice was also resistant to DNase I, suggesting that although HU facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second-strand DNA synthesis and transgene expression. Thus, the identification of a yet another obstacle — virus uncoating — has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.